Antioxidant Capacity, Phytochemicals, Minerals, and Chemical Pollutants in Worker Honey Bee (Apis mellifera L.) Broods from Northern Thailand: A Safe and Sustainable Food Source

Honey bee brood (HBB) (Apis mellifera L.), a traditional protein source, has been studied for its nutritional value, but bio-functional properties and safety concerns have not been verified. This study examined the Antioxidant capacity, phytochemicals, minerals, and chemical pollutants in worker broods from several apiaries in Northern Thailand. HBB samples were lyophilized to evaluate antioxidant capacity using ABTS, DPPH, and FRAP assays, tests with water, and 70% ethanol extracts. Phytochemicals were identified using LC-QTOF-MS; pollutants were analyzed chromatographically, and minerals were determined using ICP-OES. The results showed that the evaluated antioxidant capacity of the ethanol extracts included DPPH 2.04–3.37 mg/mL, ABTS 21.22–33.91 mg/mL, and FRAP 50.07–104.15 mg AAE/100 g dry weight. Water extracts had outstanding antioxidant activities except for ABTS, with DPPH 10.67–84.97 mg/mL, ABTS 9.25–13.54 mg/mL, and FRAP 57.66–177.32 mgAAE/100 g dry weight. Total phenolics and flavonoids in ethanol extracts ranged from 488.95–508.87 GAE/100 g to 4.7–12.98 mg QE/g dry weight, respectively. Thirteen phytochemicals were detected and contained adequate mineral contents in the HBBs from different locations found, which were K, Ca, Mg, and Na, and no heavy metals or pollutants exceeded safe levels. These results imply that HBB from different apiaries in Northern Thailand is a nutritious food source with considerable antioxidants and a safe and sustainable food source.


Introduction
Honey bees (Apis mellifera L.), commonly known as Western honey bees, are indeed widely recognized and extensively utilized in beekeeping and honey production [1].Since time immemorial, human civilization has shared a profound and enduring connection with honey bees, treasuring their precious gift of honey not only as a delectable food source but also as a medicine, a testament to the enduring harmony between mankind and these remarkable creatures [2].Until recent times, the focus of our interaction with honey bees Foods 2024, 13,1998 2 of 14 has predominantly centered on the extraction of various hive products, including honey, wax, propolis, royal jelly, and bee venom [3].Surprisingly, the bees themselves, although consumed by people in Asia and Africa in various instances throughout history, have received far less recognition [1,[4][5][6][7][8][9].Nevertheless, in recent years, there has been a growing interest in honey bees as a potential food and feed source [10].The scientific literature now exists that explores their nutritional value, shedding light on the remarkable benefits they offer [11].
Honey bee brood (HBB) plays a pivotal role in global ecosystems by serving as pollinators for a diverse array of crops and wild plants [3].The health and well-being of honey bee colonies are critical not only for honey production but also for maintaining biodiversity and ensuring food security [12][13][14][15].Understanding the nutritional composition of HBB is essential, as it directly influences the overall vitality and resilience of the colony [16].Honey bee brood's nutritional composition is of interest for human consumption for several reasons.Notably, it has been shown that HBBs have a high nutritional value.They have the greatest protein content, at 50.4%, followed by carbs at 22.1%, and fat at 20.5% [17,18].Furthermore, essential minerals and vitamins abound in insects, with varying amounts contingent upon the species and age of the bug compared to adults.The larval stage-such as silkworms, which are said to be another protein-rich meal that has a high mineral content and a very high vitamin A content-is 273.99 mg/100 g of dry weight, which offers 400-500 kcal of energy per 100 g of dry weight [17,18].
The antioxidant capacity in HBB is a crucial aspect influenced by various factors.Studies have shown that honey bee products like drone brood homogenate exhibit significant antioxidant properties, with freeze-dried and sterilized HBB showing increased radical-scavenging activity and total reducing capacity [19,20].Antioxidant capacity, a key parameter in assessing cellular defense mechanisms, is crucial for protecting HBB from oxidative stress [21].Phytochemicals derived from the diverse floral sources that bees forage on not only provide essential nutrients but may also confer additional health benefits [22].Furthermore, the antioxidant protection in honey bees varies with age, with older bees displaying higher total antioxidant capacity (TAC) and being less susceptible to pesticide toxicity, highlighting the importance of age-related antioxidant defenses in honey bees [23].HBB is rich in minerals such as potassium, magnesium, calcium, and phosphorus, making them a valuable source of essential nutrients [11,17,18].The mineral content of HBB is vital for proper growth, development, and metabolic functions, while the presence of chemical pollutants raises concerns about potential threats to bee health and, consequently, the ecosystems they support [24].However, there has been relatively limited effort to explore the bio-functional compounds within HBBs.It is well-known that honey bees frequently forage in agricultural areas, heightening the risk of chemical pollutant accumulation, such as pesticides, within their hives [25].This could result in contamination of adult bees, beeswax, and broods, thereby posing potential health risks for consumers of bee products.
The local population in Thailand acknowledges bee brood consumption, but it remains a somewhat niche aspect of their culinary tradition rather than being part of the mainstream food culture.There is a pressing need to investigate the bioactive components, mineral content, and chemical contaminants present in HBBs.While recent efforts have been made to investigate the nutritional potential of honey bees, issues such as bio-functional properties and safety concerns remain unresolved.This study investigated the antioxidant capacity, phytochemicals, minerals, heavy metal levels, and chemical contaminants to enhance understanding of the quality and safety issues associated with honey bee worker broods in Northern Thailand.

HBB Samples
Five samples of HBB were collected from apiaries or honey bee farms in Northern Thailand, including Nong Muang Kai District, Song District, Sung Men District Phrae Province, and San Sai District Chiang Mai Province (Figure 1) in November 2022.The samples were separated from honeycombs by being passed under hot steam.This method Foods 2024, 13, 1998 3 of 14 ensured that the brood was gently and effectively removed without damaging the samples.Once separated, the samples were immediately frozen at −20 • C until further analysis.Prior to analysis, these samples were freeze-dried to complete dryness using the SP VirTis Genesis Pilot Freeze Dryer (SP Stone Ridge, NY 12484, USA).The brood consisted primarily of pupae, with larvae making up less than 10% of the total.The resulting dried samples were ground into a powder and stored in a −20 • C freezer for further analysis.The water content of the bee brood samples fell within the range of 68.75-77.51%(Table 1).

HBB Samples
Five samples of HBB were collected from apiaries or honey bee farms in Northern Thailand, including Nong Muang Kai District, Song District, Sung Men District Phrae Province, and San Sai District Chiang Mai Province (Figure 1) in November 2022.The samples were separated from honeycombs by being passed under hot steam.This method ensured that the brood was gently and effectively removed without damaging the samples.Once separated, the samples were immediately frozen at −20 °C until further analysis.Prior to analysis, these samples were freeze-dried to complete dryness using the SP VirTis Genesis Pilot Freeze Dryer (SP Stone Ridge, NY 12484, USA).The brood consisted primarily of pupae, with larvae making up less than 10% of the total.The resulting dried samples were ground into a powder and stored in a −20 °C freezer for further analysis.The water content of the bee brood samples fell within the range of 68.75-77.51%(Table 1).One g of lyophilized HBB samples was subjected to an extraction process using 10 mL of deionized water and 70% ethanol.The samples were shaken at 2500 rpm for 5 min at room temperature.Subsequently, the samples were filtered using Whatman No. 1 filter paper and stored in a refrigerator at 4 °C before further analysis.One g of lyophilized HBB samples was subjected to an extraction process using 10 mL of deionized water and 70% ethanol.The samples were shaken at 2500 rpm for 5 min at room temperature.Subsequently, the samples were filtered using Whatman No. 1 filter paper and stored in a refrigerator at 4 • C before further analysis.

DPPH Radical-Scavenging Activity
The DPPH radical-scavenging activity of the HBB extract was conducted following the method described by Siok Peng Kek et al. [26].This study employed a 1,1-diphenyl-2-picryhydrazyl (DPPH) assay for analysis.A total of 100 µL of HBB extract solution was mixed with 100 µL of DPPH working solution in the 96-well plate.Subsequently, the mixture was incubated in the dark for 30 min.The absorbance at 517 nm was then determined using a microtiter plate reader (SPECTRO Star Nano) to calculate the percentage of DPPH-free radical-scavenging activity, and DPPH was determined by applying a calibration curve range of 1.562-100 µg/mL.The DPPH free radicalscavenging activity was calculated using the formula DPPH = [A control − A sample ]/A control × 100, where A control and A sample were absorbance readings.The IC50 value, indicating the sample concentration needed to inhibit 50% of DPPH free radicals, was determined from the curve relating DPPH scavenging activity percentages to sample concentration.

ABTS Radical-Scavenging Activity
The ABTS radical-scavenging activity was carried out following the experimental procedure outlined by Arnao et al. [26].A stable radical, ABTS reagent, was employed in an aqueous solution containing 2,2-casino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, with Trolox serving as a reference.To a 96-well plate containing 190 µL of ABTS working solution, 10 µL of HBB extract solution was added.After incubating in the absence of light for 10 min, absorbance was measured at a wavelength of 734 nm, and a calibration curve range of 3.125-100 µg/mL was used to determine ABTS.The ABTS radical-scavenging effect was calculated using the following formula: ABTS radical-scavenging effect % = ((Ab − Aa)/Ab) × 100, where Ab represents the absorbance of the ABTS radical with methanol, and Aa is the absorbance of ABTS radicals in the sample extract.

Ferric Ion Reducing Antioxidant Power Assay (FRAP)
The FRAP test was performed following the experimental procedure outlined by Kek et al. [27].The FRAP reagent, a solution, was formulated by mixing 2.5 mL of a 10 mM TPTZ (2,4,6-tripyridyl-s-trizine) solution in 40 mM HCl, 2.5 mL of a 20 mM ferric chloride (FeCl 3 ) solution, and 25 mL of a 300 mM acetate buffer (pH of 3.6).Ten microliters (10 µL) of the diluted HBB extract solution was added with 190 µL of FRAP reagent in a 96-well plate.After 30 min in the absence of light, the sample's absorbance was measured at a wavelength of 593 nm using a microtiter plate reader, and FRAP was determined by applying a calibration curve range of 1.562-100 µg/mL.The quantification of the outcome was expressed as milligrams of ascorbic acid equivalent per 100 g of HBB extract (mg AAE/100 g).

Total Phenolic Content (TCP)
The total phenolic content was determined using the Folin-Ciocalteu method, adapted with minor modifications from the procedure outlined by Dewanto et al. [28].Gallic acid (Fluka, 9470 Buchs, Switzerland) served as the standard substance in this methodology.Thirteen microliters (13 µL) of the HBB extract solution sample was combined with 13 µL of the Folin-Ciocalteau solution (MERCK, 82024, Taufkirchen, Germany), diluted with 10 µL of distilled water.After a 6-min interval, a solution consisting of 100 µL of distilled water and 125 µL of 7% Na 2 CO 3 was added.Subsequently, the mixture underwent a 90-min incubation at room temperature.Absorbance was subsequently determined using a microplate reader (SPECTRO Star Nano) at a wavelength of 760 nm.The phenolic concentration was determined by applying a calibration curve range of 20-1000 µg/mL, and the results were expressed in gallic acid equivalents per gram of sample (mg GAE/100 g sample).

Total Flavonoid Content (TFC)
The determination of the total flavonoid content followed the method outlined by Miliauskas et al. [29].Twenty-five microliters (25 µL) of HBB extraction solution was mixed with 8 µL of 7% NaNO 2 and 12.5 µL of distilled water and vigorously stirred.The solution was allowed to stand at room temperature for five minutes.Subsequently, 15 µL of a 10% AlCl 3 solution was added, and the components were left at room temperature for another five minutes.Further, 50 µL of 1M NaOH solution and 28 µL of distilled water were added.After thorough mixing, the mixture was kept at room temperature for an additional five minutes.Absorbance was measured at 510 nm using a SPECTRO Star Nano microplate reader.The quantification of flavonoids was accomplished through the utilization of calibration curves range 10-500 µg/mL.The results were measured and expressed as quercetin equivalents per gram of sample (mg QE/g sample).

LC-Q-TOF/MS Identification of Phytochemicals
Phytochemical analysis was conducted using an ultra-performance liquid chromatography system coupled with a quadrupole time-of-flight mass spectrometer (Q-TOF-MS).Agilent 1290 Infinity II series coupled to the 6546 instruments (Agilent Tech., Santa Clara, CA, USA) in both positive and negative ionization modes, as previously described in standard methodologies [30,31].The quantification of phytochemicals was performed through the utilization of the Medlin Library.

Mineral Analysis 2.4.1. Sample Preparation
The dried HBB samples, weighing 0.2 g, were placed in a 100 mL Teflon vessel.The microwave extraction technique was employed for sample extraction.Nine milliliters of nitric acid and 1.0 mL of hydrogen peroxide were added to the samples, which were then placed in a 210 • C oven at 18,000 watts.The microwave extraction process lasted for 35 min, and the sample was allowed to cool to room temperature.The resulting mixture was combined with 1 mL of distilled water, vortex-mixed, and filtrated using a 0.2-µm syringe filter prior to analysis in inductive-coupled plasma optical emission spectrometry (ICP-OES, Agilent 5800 series, Stevens Creek Blvd., Santa Clara, CA, USA).

ICP-OES Determination of Minerals
The mineral composition of HBB samples was determined according to the study of Kawichai et al. [32] with certain modifications, which are detailed as follows.

Organophosphate and Synthetic Pyrethroid Insecticide Analysis
The process of extracting HBB samples involved techniques adapted from previous studies with slight modifications [33].Standard stock solutions of organophosphates (OPs) and pyrethroids (Pys) were employed.The OPs included methamidophos, mevinphos, diazinon, dicrotophos, monocrotophos, dimethoate, pirimiphos-methyl, chlorpyrifos, parathion-methyl, malathion, fenitrothion, prothiophos, methidathion, profenphos, ethion, triazophos, EPN, and azinphos-ethyl, while the Pys encompassed enpropathrin, lambda-cyhalothrin, permethrin, cyfluthrin, cypermethrin, fenvalerate, esfenvalerate, and dethamethrin.These standards were thoroughly prepared in ethyl acetate and methanol, each at a concentration of 1000 mg/L, and stored at −20 • C in the refrigerator.Prior to analysis with GC-FPD and GC-ECD, working standards for organophosphate and synthetic pyrethroid insecticides were prepared by diluting the stock standards with ethyl acetate.The calibration curves of all pesticides range from 0.05-0.60mg/kg.
The HBB sample was homogenized, weighed at 2.5 g into 50 mL centrifuge tubes, and spiked with 100 µL of working standards and internal standards.Ten milliliters of deionized water (DI) and 10 mL of acetonitrile (ACN) were added, and the sample was shaken for two minutes.The tubes were centrifuged at 3000 rpm for 5 min.The supernatant was transferred to a 15 mL centrifuge tube containing 4 g MgSO 4 , 1 g NaCl, and 1 g Na 3 C 6 H 5 O 7 and shaken for 15 min.The slurry was then centrifuged at 3000 rpm for 5 min.The 5 mL of supernatant was transferred to a 15 mL centrifuge tube containing EMR lipid sorbent and immediately vortexed to disperse, then centrifuged at 3000 rpm for 5 min.The upper I layer was filtrated through a 0.22 µm syringe filter and transferred to sample vials to be ready for analysis of pesticide residue by GC-FPD and GC-ECD.The extract samples were analyzed using GC chromatography for pesticide residue organophosphate group separation by GC-FPD Model 7890B (columns used DB-1701, 0.25 mm, 30 m length, 0.25 µm film thickness, Agilent J & W column, Agilent Technologies, Santa Clara, CA, USA), pyrethroid group, and organochlorine using GC-ECD Model 7890B (column using HP5, 0.25 mm × ID × 30 m length × 0.25 µm film thickness, Agilent J & W column, Agilent Technologies, USA).The data were analyzed by OpenLab Chem Station software version C.01.07 (Agilent Technologies, Santa Clara, CA, USA).

Organochlorine Pesticide Analysis (OCs)
The bee brood sample was extracted by the method described earlier [34].The Ocs pesticides, i.e., beta-HCH, heptachlor, endosulfan, o,p ′ -DDT, p,p ′ -DDT, o,p ′ -DDE, p,p ′ -DDE, and p,p ′ -DDD, were analyzed.The 2.5 g of homogenized sample was extracted with 15 mL of acetonitrile in 1% acetic acid and shaken vigorously for 1 min.Then, 4 g of MgSO 4 , 1 g of NaCl, and 1.5 g of C 2 H 3 NaO 2 were added and vortexed for 5 min.The solutions were centrifuged at 3000 rpm for 5 min, and then 5 mL of the extract was transferred into a 15 mL centrifuge tube containing EMR Lipid.The extracted sample was vortexed for 1 min and centrifuged at 3000 rpm for 5 min.The supernatant was pipetted into 2 mL of water to evaporate to dryness.The extracted sample was added to 1 mL of methanol and filtrated through a PTFE syringe filter prior to analysis by GC-ECD.The calibration curves range from 0.05-0.60mg/kg.

Statistical Analysis
Each experiment was performed three times, and the results were expressed as mean ± standard deviation.To examine the differences, one-way paired-samples t-test and one-way ANOVA, followed by DMRT (p < 0.05), were performed using IBM SPSS Statistics version 29.0.1.0.

Antioxidant Activity
Table 2 presents the antioxidant activities of HBB samples obtained from different farms situated in Northern Thailand.The findings indicated that all the samples examined exhibited antioxidant activity, with a significant degree of variability.The antioxidant capacity of ethanol-extracted samples was demonstrated by IC 50 of DPPH (2.04-3.37)mg/mL, ABTS (21.22-33.91)mg/mL, Foods 2024, 13, 1998 7 of 14 and FRAP (50.07-104.15)mg AAE/100 g dry weight.The water-extracted samples demonstrated an IC 50 of DPPH (10.67-84.97)mg/mL, ABTS (9.25-13.54)mg/mL, and FRAP (57.66-177.32)mg AAE/100 g dry weight.Except for ABTS HBB, samples extracted with water exhibited superior antioxidant properties, possibly attributed to the polar compounds extracted in an aqueous solution.

Total Phenolic Content (TPC) and Total Flavonoids Content (TFC)
The Total Phenolic Content (TPC) and Total Flavonoid Content (TFC) of the HBBs are depicted in Table 3.The TPC and TFC of 70% ethanol extracted were within the range of 488.95-508.87mg GAE/100 g dry weight and 4.71-12.98mg QE/g dry weight, respectively.The TPC and TFC of DI water extracted range from 496.06-518.74mg GAE/100 g dry weight and 5.32-21.60mg QE/g dry weight, respectively.In both Total Phenolic Content (TPC) and Total Flavonoid Content (TFC) estimations, the water-extracted samples consistently displayed higher values, aligning with the increased antioxidant levels observed in these specific samples.

Phytochemical Analysis
This investigation confirmed the presence of phytoconstituents in the HBB extract, considering the molecular formula observed in m/z values, exact mass, and retention time.A detailed account of each annotated compound is presented in Table 4. Thirteen phenolic and flavonoid compounds, including 5Z-caffeoylquinic acid, gallic acid, protocatechuic acid-3-glucoside, protocatechuic acid, ellagic acid, rutin, orientin, epicatechin, vitexin, quercetin, pinobanksin, kaempferol, and caffeic acid, were identified.Five compounds, including protocatechuic acid, orientin, pinobanksin, kaempferol, and caffeic acid, were found in all the samples.Notably, all detected compounds exhibited a matching score, which is the value representing the match quality between the observed mass spectrum and the reference spectrum in a database exceeding 90%.RT is short for "retention time" here (+) = presence of constituents; (−) = absence of constituents.
On the other hand, heavy metals, including Al, As, B, Ba, Ti, Cr, Cu, Mo, Ni, Pb, Sb, and Se, were detected, but their concentration was relatively low, as represented in Table 6.The results are presented as mean values ± standard deviation.Different superscript letters within the same row indicate a significant difference in heavy metals between locations (p ≤ 0.05).ND-Not detected.

Chemical Pollutants Analysis
The findings indicated the absence of any traces of harmful chemicals in all samples, even when considering the relatively high limit of detection (LOD) for the analysis OPs, 0.001-0.008mg/kg; PYs, 0.001-0.007mg/kg; OC, 0.002-0.007and PAHs, 1.00-51.78mg/kg.The linearity of the calibration curve was evaluated by investigating the correlation coefficient (R 2 ) of the calibration curves.The r values were higher than 0.998 for all the analytes, as shown in Table 7. None of the polluting chemicals were detected in the bee brood samples.The antioxidant potential of HBBs suggests that antioxidants play a crucial role in reducing the production of harmful free radicals during food digestion, thus diminishing the risk of disease [35].The DPPH and FRAP assays revealed higher values for extracts obtained with DI water compared to those extracted with 70% EtOH, whereas the ABTS radical-scavenging assay demonstrated greater efficacy for extracts obtained with 70% EtOH compared to those with DI water.The brood exhibits strong radical-scavenging activity in DPPH and ABTS tests, indicating its effectiveness in removing free radicals, as supported by prior research [35,36].This trend is also evident in TPC and TFC, showing comparable levels in DI water and 70% EtOH extracts.According to the results obtained by Sawicki et al. [37], the TPC and TFC content in HBB showed high levels when compared with bee pollen, bee bread, and beeswax.Additionally, Lin et al. [6] reported that bee broods, especially drone broods, are rich in antioxidants such as ascorbic acid, coenzyme Q10, and all-trans-retinol.Phenolic and flavonoid compounds, known for their antioxidant properties, play a vital role.Some flavonoids exhibit metal-chelating properties crucial for inhibiting reactive oxygen species (ROS) formation and inhibiting enzymes like protein kinase and xanthine oxidase [37].Moreover, flavonoids serve various functions in plants, including photoreception, antimicrobial activity, visual attraction, and herbivore repellency [38].Carbohydrates with unbound aldehyde or ketone groups can function as reducing agents [39].Excessive sugar intake is linked to unhealthy weight gain and Foods 2024, 13, 1998 10 of 14 diabetes onset, emphasizing the importance of carbohydrate restriction for maintaining a healthy weight [40,41].The use of HBBs should not pose an elevated risk of obesity and associated complications for both children and adults [42].

Phytochemicals
Insects are gaining recognition as promising sources of health-boosting bioactive compounds, positioning them as potential functional foods [43].Edible insects, such as silkworms, grasshoppers, mealworms, giant worms, and HBBs, are known for their antioxidant properties and potential role in preventing oxidative stress [44].Torres-Castillo Certain et al. [45] found that edible insects contained bioactive compounds like β-carotene as well as flavonoids like kaempferol and quercetin.These bioactive substances are metabolized or absorbed by insects from the host plants that produce them as secondary metabolites, according to the results of Hussain et al. [44].Phenolics, recognized as the most potent antiradicalcontaining compounds, activate enzymes that safeguard cells against damage related to oxidative stress [46].Through UHPLC-QTOF-MS analysis, phytochemical compounds were identified in the HBB ethanolic extract, including gallic acid, caffeic acid, protocatechuic acids, kaempferol, quercetin, and rutin.Notably, additional compounds, such as caffeine, kaempferol, pi-nobanksin, quercetin, orientin, vitexin, rutin, caffeoylquinic acid, and proto-catechuic acid, were detected, aligning with previous studies by Sawicki et al. and Silva et al. [37,47].This underscores the diverse array of beneficial compounds present in HBB, further highlighting its potential as a valuable source of bioactive components.

Mineral Content
The nutritional composition of HBB exhibits variation across different developmental stages yet consistently maintains high levels of protein, fat, carbohydrates, and minerals [37].These minerals, linked to diverse health benefits such as bone health and optimal metabolic function in the human body, are noteworthy [47].The brood displays elevated concentrations of potassium, with a comparatively lower concentration of sodium.High potassium and low salt content could have positive nutritional effects, especially for the segment of the population that suffers from hypertension; our results agree with those of Solayman et al. [48].Regarding trace elements, this study indicates that the HBB possesses significant quantities of iron, zinc, and copper while revealing a lower amount of manganese, and the findings are in agreement with previous studies [1,[4][5][6][8][9][10].Calcium is crucial for bone and dental health, heart rhythm, muscle function, nerve activity, hormone regulation, and blood pressure [49].Adequate daily calcium intake is crucial for overall well-being and can be achieved through a diverse diet incorporating calcium-rich foods [50].Sufficient dietary calcium is particularly important for supporting bone growth, with pregnant women requiring it for fetal bone development and postmenopausal women prioritizing calcium intake to reduce the risk of osteoporosis [34].
Iron deficiency, the primary cause of anemia globally, arises from reduced iron intake, blood loss, or impaired iron absorption.This deficiency, known as iron deficiency anemia (IDA), is a significant global health issue affecting millions of people, especially women and children, in developing and underdeveloped regions [25].
Zinc is essential for various metabolic processes, including protein synthesis, transcription, and DNA replication [51].Zinc deficiency affects human health and can manifest in symptoms like stunted growth, appetite loss, and impaired smell and taste [44,52].Severe zinc deficiency can impact various organ systems, including the immune, gastrointestinal, central nervous, skeletal, and reproductive systems [53].Assuming favorable bioavailability, the inclusion of HBB in one's diet could be anticipated to provide essential minerals of nutritional significance at noteworthy levels.Contrastingly, the current study indicates that HBB samples from various apiaries do not contain any heavy metals exceeding safe levels.

Food Safety
Pesticide contamination in honey bee larvae poses a significant concern, as it has the potential to adversely impact bee health, leading to increased mortality [54].Medina and Ravoet et al. and Ravoet et al. [55,56] found pesticide residues commonly found in wax combs, often characterized by their lipophilic nature or dissolved in oil-based carriers.These substances may be introduced into the hive through contaminated nectar and pollen by honey bees or by beekeepers seeking to control parasites, according to the results of Karazafiris [57].This contamination can result in the transfer of pesticides into various food resources, including honey, beebread, and bee broods [57,58].Beeswax, utilized in the food, cosmetics, and pharmaceutical industries, may expose humans to pesticides [51].By identifying environmental chemicals, such as pesticides and polycyclic aromatic hydrocarbons (PAHs), as potential contaminants, as reported by Arjin et al. and Amekawa et al. [59,60], this study did not determine if the samples surpassed the limit of detection (LOD) for hazardous compounds, indicating the absence of such substances in the examined HBB samples.It is imperative to establish chemical safety for bee broods to gain consumer acceptance, as the presence of harmful elements, pesticide residues, or other environmental contaminants can diminish their nutritional value and nutritional benefits [61].

Conclusions
This study found that HBB was a promising and sustainable food source, given its antioxidant properties, an abundance of phenolic compounds, and rich mineral content, potentially offering various health benefits.Hydrophilic antioxidants play a substantial role in antioxidant activity levels, which can vary significantly depending on the bee's diet, the time of year, and the specific environmental conditions, so the limitations of this study did not account for the potential seasonal variations that might affect the composition of HBB, including variances in the levels of minerals, such as sodium, potassium, etc., in the HBB, which are probably influenced by their environments, ecological traits, and the diverse plant sources they feed on.The current research indicates that HBBs from different apiaries are free of harmful chemicals and contaminants, confirming their safety for consumption.Embracing HBB as a food source contributes to food and nutrition security by presenting a locally available, natural, and sustainable option that complements diets and enhances nutritional value.Future studies should include samples collected across different seasons to understand how seasonal changes impact the nutritional and chemical characteristics of HBB and how awareness of HBB consumption has the potential to yield health benefits.

Figure 1 .
Figure 1.Geographical positioning of the HBB collection site in the northern region of Thailand.

Figure 1 .
Figure 1.Geographical positioning of the HBB collection site in the northern region of Thailand.

Table 1 .
Geographical locations of bee brood samples collection and their water content (%).

Table 1 .
Geographical locations of bee brood samples collection and their water content (%).

Table 2 .
Antioxidant activity (DPPH, ABTS, and FRAP) in HBB extracted samples of different honey bee farm locations.

Table 3 .
Total phenolic content (TPC) and total flavonoid content (TFC) in HBB-extracted samples of different honey bee farm locations.

Table 4 .
Phytochemical identification of different honey bee farm locations by LC-Q-TOF/MS.

Table 5 .
Mineral content (mg/100 g dry matter basis) in the HBB samples from different honey bee farm locations.

NT-1 HB-NT-2 HB-NT-3 HB-NT-4 HB-NT-5
The results are presented as mean values ± standard deviation.Different superscript letters within the same row indicate a significant difference in mineral content between locations (p ≤ 0.05).ND-Not detected.

Table 6 .
Heavy metals (mg/100 g dry matter basis) in the HBB samples from different honey bee farm locations.

Table 7 .
Limit of detection, working standard range, and correlation coefficient of the calibration curve for the chemical pollutants analysis.